The four-helix bundle structural motifs is found in a variety of proteins that perform a wide range of functions. This proposal focuses upon determining the basis of the structure and stability of this motif, using Rop (Rom) as a model system. Rop is a homo-dimer of two helix-loop-helix monomers, each of which is 63 amino acids long. The function of Rop is to bind to a specific RNA complex. As a consequence of this protein-RNA interaction, plasmid copy number in ColE1 plasmids is regulated. The x- ray crystal structure of Rop is known at 1.7A resolution, its 1H NMR spectrum has been assigned and its small size is convenient for molecular modelling. We propose to take wild-type Rop and by systematic re-design and simplication to determine the structural features that are essential for a stably folded and functional protein. The three classes of proteins that we will study are: 1) Proteins with re-packed interiors. Rop's internal packing can be interpreted as eight receptions of a characteristic four amino acid "layer". The layers are perpendicular to the long axis of the bundle. Our aim is to delineate which layer redesigns will generate a correctly and stably problem and will allow us to approach the repacking of the entire protein in a systematic fashion. 2) Role of the connecting loops. The role connecting loops play in directing protein folding or i influencing protein stability is not will understood. We will investigate how increasing loop length affects protein stability and the kinetics of protein folding are affected as loop length and flexibility are systematically varied. 3) Identification of the residues involved in RNA recognition. Rop provides us with a rare opportunity to understand RNA recognition by a small protein of known structure. We will use site-directed mutagenesis to identify which residues are involved in RNA binding and will determine the effect of specific mutations on the energetics of protein-RNA complex formation. This data will be interpreted in the context of structural information on the complex that comes from our collaborator, Prof. D. Crothers, NMR studies. The techniques that will be used to characterize the proteins include CD, 1- and 2-dimensional NMR and x-ray crystallography for structural characterizations; CD, Fluorescence and Calorimetry to monitor the unfolding transition and to determine the energetics of protein folding; gel-shift assays and hydroxy-radical footprinting to determine the energetics of protein; RNA interactions and to compare the details of different protein-RNA complexes.